Drug metabolism activity is a Critical Intrinsic Selector for Hepatocytes to elongate cell lifespan by using a cell-killing antibiotic

The liver plays many important roles in homeostasis, including drug detoxification, metabolism, and bile production. Hepatocytes, which are the main constituent cells of the liver, play an important role in the pathogenesis of liver diseases, identification of candidate compounds in drug discovery research, pharmacokinetic studies, and toxicity evaluation. Human hepatocytes are, however, difficult to grow in normal in vitro culture systems, making it difficult to secure cell numbers. As an alternative, evaluation systems using animal models and hepatocellular carcinoma cells have been established, but interspecies and interracial differences and low hepatic function have been pointed out as problems. Therefore, there is still a need for a highly stable method to prepare human hepatocytes with sufficient functionality. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of the functionality of human hepatocytes to stably supply human hepatocytes. In the established culture system, the stable proliferation of human hepatocytes was achieved by co-culturing hepatocytes with mouse fetal fibroblasts to dedifferentiate them into hepatic progenitor-like cells. Furthermore, we succeeded in purifying human hepatocytes by puromycin with a rapid cytocidal effect and proliferating them to over 30 population doublings for more than 200 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytotoxic antibiotics because of enhanced drug-metabolizing activity. These results show that the above culture system enables simple and efficient hepatocyte proliferation, and is considered to be an effective method for stable supply of hepatocytes and significant cost reduction in drug discovery research.